Before scientists study a protein and its properties, the protein first needs to be purified or extracted – which is essential in protein research. Some proteins need to be studied separately, and some proteins have unnecessary impurities that need to be removed for accurate results. Protein purification is essential in protein research. There are several techniques of purifying a protein, based on its size, charge, solubility, and binding similarities.

Firstly, how do biochemists recognize the desired protein in a mixture? They use a test, called an assay, where they choose a specific reaction for a particular protein, mix it, and observe if the mixture changes. If it does, then it means the desired protein is present and needs to be purified. It is very difficult to determine an effective assay because they need to react with the protein of interest but nothing else in the mixture. Once the protein is located, biochemists break the cells open to extract it. They grind the cells up, then freeze and melt them. After that, they need to disrupt the cell membrane and pull the protein apart. The most effective way to disrupt a cell is by using rapid pressure changes.

Salting Out

In certain conditions, proteins can be more or less soluble. In order for this method to work, we need to have a salt that is very soluble. (NH₄)₂SO₄ (ammonium sulfate) is usually used for this purification method because it is very soluble and works with proteins very well. The salt concentration for each protein to precipitate is different. Therefore, we can separate one protein from another, using the salting out method. 

Dialysis

Dialysis is the method used to separate particles in a liquid, based on their size. For this technique, we use a cellulose membrane with pores. Smaller ions and molecules travel right through the pores, staying in the solution outside of the membrane. Bigger ions and molecules stick inside of the membrane because they are too big to go through the pores. Dialysis works best to separate small molecules from a protein. However, it is not effective for separating one protein from another.

Gel-Filtration Chromatography

This is a technique used to separate two different proteins based on their size. For this method, we need to fill a column with insoluble porous beads. The column used is usually long and thin because it speeds up the whole process. Small molecules can enter the beads while big molecules cannot fit into them and have to find a way out in between to pass through. Small proteins will take a longer time because they will go through the beads. Larger proteins will exit first because they will go around the beads and take less time.

Ion-Exchange Chromatography

This is a method used to separate two different proteins based on their charge. At a particular pH, if proteins have different charges, they can be separated. For this technique, we again use a column and fill it with beads that are either negatively or positively charged. If the beads are positively charged, then a protein that is also positively charged will just flow through the beads. However, a protein that is negatively charged will stick to the beads. To get them off the beads, the pH of the solution is changed, so the protein that is negatively charged will change and come off.

Affinity Chromatography

This is a technique used to purify proteins based on their tendency to bind to specific chemical groups. It is so powerful that sometimes scientists can purify proteins in one step. It requires a column filled with specific molecules or beads that particular proteins can attach to. Target molecules bind to beads while other molecules are washed through. Then, a highly concentrated elution buffer (major solvent) is used to break the bond between the beads and target molecules.
There is another protein purification technique called gel electrophoresis (a method used to separate proteins based on charge by using a specific gel). However, it will not be very helpful for purifying proteins from scratch. It is a long process, and there are other techniques that are more effective.